The Relation Of Polyamine Metabolism And Akt Signal Pathway In Tumor Cells

Objective Polyamines are alkyl amine molecules with positive charge and exist in allcells. Studies have shown that the rapid proliferation of tumor cells is highly dependent onthe intracellular levels of polyamines. Reducing polyamine content whithin tumor cells bypolyamine depletion agents, such as DFMO, can effectively inhibit tumor cell proliferation.As a specific inhibitor of ornithine decarboxylase (a rate-limiting enzyme in polyaminesynthetic pathway), DFMO can inhibit tumor cell proliferation and induce cell apoptosis.However, high-dose DFMO was required for this anti-tumor effect. And also the sideeffects arising from this high-dose, DFMO greatly limited its useless in the clinical trial ofanti-tumor therapy. Recently it has been reported that DFMO treatment could activate thePI3K/Akt signaling pathway which could promote cell proliferation and inhibit cellapoptosis. We speculate that DFMO-induced activation of PI3K/Akt signaling pathwaywill antagonize DFMO’s effects in inhibiting cell proliferation and inducing apoptosis andthis is why the high-dose DFMO is need to inhibit tumor cell proliferation. Therefore,blocking PI3K/Akt signaling pathway will be able to greatly improve the DFMO’santi-tumor activity and reduce its drug dosage and side effects when used in the clinic.Methods (1) HPLC was used to determine the effect of DFMO on polyamine contentand western blotting assay was used to evaluate the impact of DFMO on Aktphosphorylation level in A549cells;(2) Constructing a eukaryotic expression plasmidwhich contains the dominant negative mutant Akt;(3) Reducing the level of Aktphosphorylation in A549cells by using PI3K/Akt kinase inhibitor LY294002ortransfecting cells with dominant negative mutant Akt and then analyzing its effect on thecellular polyamine content.(4) Treating A549cells with DFMO and PI3K/Akt kinaseinhibitor together and then evaluating its synergistic effect on inhibiting tumor cellproliferation and inducing apoptosis.Results (1) DFMO can significantly reduce the content of putrescine, spermidine,spermine in A549cells;(2) DFMO treatment had no significant impacted on the total Aktprotein level and Akt Thr308phosphorylation, but obviously increased the phosphorylationof Akt Ser473. Supplementing putrescine, spermidine, spermine can reverse DFMO-induced Akt Ser473phosphorylation;(3) Wild-type and dominant negative mutant Aktwere cloned and inserted into the eukaryotic expression plasmid successfully;(4) Both thePI3K/Akt kinase inhibitor LY294002and dominant negative mutant Akt could inhibit Akt Ser473phosphorylation and, in the same time, significantly reduce polyamine content inA549cells;(5) Blocking Akt phosphorylation could promote DFMO’s ability in inhibitingproliferation and inducing apoptosis in A549cells.Conclusion Depleting polyamine by DFMO can up-regulate the level of Akt-Ser473phosphorylation in A549human lung cancer line. Inhibiting PI3K/Akt signaling pathwayalone may also reduce intracellular polyamine content, suggesting existence of a functionalmutual restraint relationship between polyamine metabolism and PI3K/Akt signalingpathway. Combined administration with PI3K/Akt inhibitor can significantly promoteDFMO’s ability in inhibiting proliferation and inducing apoptosis in A549cells.